The secretion of penicillinase by Bacillus licheniformis and of the glycoprotein invertase by Saccharomyces yeasts are studied to clarify the mechanism of enzyme secretion in microorganisms and its relation to the process in animal cells. We will emphasize the production of penicillinase in vitro, first using preparations from Escherichia coli to obtain the initial gene product, and then shifting to a system from B. licheniformis to follow the steps in formation of the phospholipoprotein penicillinase present in the cell membrane. Special attention will also be given to the factors that affect the accessibility of this enzyme to cleavage by the penicillinase-releasing protease (PR-protease). Other possible functions for PR-protease will be investigated. The biosynthesis of invertase will be examined in a new mutant that lacks the internal (small enzyme). This should facilitate identification of intermediates in the production of the glycoprotein form. Cycloheximide and tunicamycin will be employed to block the formation of the polypeptide or carbohydrate moieties.